The long term goal of this project is to determine the structure and function of the membrane proteins of the mammalian lens. A number of major proteins that may effect gap junction-mediated cell-to cell communication have now been identified in the mammalian lens fiber cell plasma membrane. These include MP20 and MP26 that are present in all regions of the lens, but are present in junctional plaques in only a small region of the lens outer cortex. Two connexin proteins connexin46 (Cx46) and connexin-related MP70 have been localized to junctional plaques throughout the outer cortex regions of lens fiber cells, while a single connexin protein connexin43 has been identified in lens epithelial cells. AU these protein are substrates for several different protein kinases, while Ca2+, plays a key role in regulating the levels of 2nd messengers that may potentially activate these kinases. The specific aims of this project are to determine both the roles of these several major lens membrane proteins, and the mechanisms regulating lens cytosolic Ca2+ concentration. This project has four major goals: (1) The organization, membrane associations, and role of MP20 in the lens fiber cell membranes will be determined using chemical modification reagents, antibodies, and single channel activity in planar lipid bilayers. (2) To determine the organization and role of Cx46 and MP70, peptide antibodies generated to appropriate cDNA derived sequences of ovine Cx46 and MP70 will be developed; the connexin kinase(s) and their substrate sequences will also be determined in this section. (3) The possible roles of the candidate junctional proteins in cell-to-cell communication will be determined in an ovine lens culture system using site-specific polyclonal antibodies, 35S to measure protein turnover, anti-sense oligonucleotides to inhibit the expression of specific proteins, and in situ hybridization to localize mRNA transcripts to specific cell types in the culture system. (4) The mechanisms responsible for elevating Ca2+ in the ovine lens cultures will be determined using the Ca2+ indicator Fura-2 and an image analysis system; the effect of a number of agents that have been proposed to induce cataract formation by elevating lens cytosolic Ca2+ will also be determined. The relationship between Ca2+ and cAMP levels in the lens will also be determined in this section, as will the hypothesis that cAMP is only elevated in the lens following an elevation of cytosolic Ca2+. These different approaches should provide a clearer understanding of the organization, expression, and roles of these major lens membrane proteins. This should lead to an improved understanding of lens fiber cell regulation, and thus, a better basis for interpreting the nature of cataract formation. The development of a system that can accurately measure cytosolic Ca2+ levels in a differentiating lens culture system should provide a valuable tool in understanding the role of Ca2+ in the development of this pathologic process.